Application Device for Inducing Cytotoxicity to Tumor Cells Via Coated Cerium Oxide Nanoparticles

ABSTRACT

Differential surface-charge-dependent localization of nanoceria in normal cells and cancer cells plays a critical role in the toxicity profile of a nanoceria particle. Engineered surface-coated cerium oxide nanoparticles with different surface charges that are positive, negative and neutral provide therapeutic results for normal and cancer cell lines. Results show that nanoceria with a positive or neutral charge enters most of the cell lines studied, while nanoceria with a negative charge internalizes mostly in the cancer cell lines. Moreover, upon entry into the cells, nanoceria is localized to different cell compartments (e.g. cytoplasm and lysosomes) depending on the nanoparticle surface charge. The internalization and subcellular localization of nanoceria plays a key role in the nanoparticle cytotoxicity profile, exhibiting significant toxicity when they localize in the lysosomes of the cancer cell lines. In contrast, minimal toxicity is observed when they localize into the cytoplasm or do not enter the cells.

This application is a divisional of U.S. Ser. No. 13/188,695 filed onJul. 22, 2011 which claims priority based on U.S. Provisional PatentApplication Ser. No. 61/366,697 filed on Jul. 22, 2010, both of whichare incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to polymer coated nanoceria particles and moreparticularly to treatment of human and animal cells wherein cellinternalization and cytotoxicity of polymer-coated nanoceria plays a keyrole in the nanoparticle cytotoxicity profile, exhibiting significanttoxicity when localized in the lysosomes of cancer cell lines. Theresearch herein is partially funded by the National Institutes of Health(NIH) under contract number CA10178.

BACKGROUND AND PRIOR ART

Nanomaterials with unique magnetic, luminescent and catalytic propertiesare being engineered for numerous biomedical applications, ranging fromimaging, diagnostics and therapy, as reported in the followingreferences: R. Weissleder, Molecular Imaging in Cancer. Science 2006,312, 1168-1171; N. L. Rosi, N. L et al. Oligonucleotide-Modified GoldNanoparticles for Intracellular Gene Regulation. Science 2006, 312,1027-1030; M. Lewin et al, Tat peptide-derivatized MagneticNanoparticles Allow In vivo Tracking and Recovery of Progenitor Cells.Nat Biotechnol 2000, 18, 410-414; S. George et al., Use of a RapidCytotoxicity Screening Approach To Engineer a Safer Zinc OxideNanoparticle through Iron Doping. ACS Nano 2010, 4, 15-29; T. A. Xia etal., Polyethyleneimine Coating Enhances the Cellular Uptake ofMesoporous Silica Nanoparticles and Allows Safe Delivery of siRNA andDNA Constructs. ACS Nano 2009, 3, 3273-3286; Y. Roiter, et al.,Interaction of Nanoparticles with Lipid Memberane. Nano Lett 2008, 8,941-944; H. Vallhov, et al., Mesoporous Silica Particles Induce SizeDependent Effects on Human Dendritic Cells. Nano Letters 2007, 7,3576-3582. However, the greatest strength of nanomaterial, which reliesprimarily on the enhanced physical and chemical characteristics thatmatter exhibits at this scale, has the potential to be its greatestliability.

Potentially harmful interactions can occur between nanomaterials andliving systems, including systems in the human body. For this reason,nanomaterials must be engineered using materials that either arenon-toxic, biocompatible and biodegradable or that have minimal and insome cases beneficial properties. An inflammatory response is aparameter that is often investigated to assess the effect thatnanomaterials have within an organism, as reported by A. E. Nel et al,in Understanding Biophysicochemical Interactions at the Nano-bioInterface. Nat Mater 2009, 8, 543-557.

For instance, recent studies have shown that titanium oxidenanoparticles, which are widely used in cosmetics and skin careproducts, can elicit an inflammatory response and the generation ofreactive oxygen species, causing DNA damage, according to B. C. Schanenet al., in Exposure to Titanium Dioxide Nanomaterials ProvokesInflammation of an In vitro Human Immune Construct. ACS Nano 2009, 3,2523-2532 and A. A. Shvedova et al., in Exposure to Carbon NanotubeMaterial: Assessment of Nanotube Cytotoxicity using Human KeratinocyteCells. J Toxicol Environ Health A 2003, 66, 1909-1926.

Also, single-walled carbon nanotubes can cause lipid peroxidation,oxidative stress, mitochondrial dysfunction and changes in cellmorphology upon in vitro incubation with keratinocytes and bronchialepithelial cells, as discussed by C. W. Lam et al., in HistopathologicalStudy of Single-Walled Carbon Nanotubes in Mice 7 and 90 days afterInstillation into the Lungs. Abstracts of Papers of the AmericanChemical Society 2003, 225, U955-U955.

Furthermore, silver nanoparticles have been found to displaysize-dependent toxicity when exposed to alveolar macrophages viainduction of oxidative stress, as reported by C. Carlson et al., inUnique Cellular Interaction of Silver Nanoparticles: Size-dependentGeneration of Reactive Oxygen Species. J Phys Chem B 2008, 112,13608-13619 and S. M. Hussain et al., in Safety Evaluation of SilverNanoparticles: Inhalation Model for Chronic Exposure. Toxicol Sci 2009,108, 223-224.

Quantum dots and fullerenes can also initiate an inflammatory responseand the generation of reactive oxygen species, as discussed by H. H.Chen et al., in Acute and Subacute Toxicity Study of Water-SolublePolyalkylsulfonated C₆₀ in Rats. Toxicologic Pathology 1998, 26,143-151; H. H. Chen et al., in Renal Effects of Water-solublePolyarylsulfonated C60 in Rats with an Acute Toxicity Study. FullereneScience and Technology 1997, 5, 1387-1396 and A. Nel et al., in ToxicPotential of Materials at the Nanolevel, Science 2006, 311, 622-627.

Cerium oxide nanoparticle (nanoceria) is a unique nanomaterial, becauseit exhibits anti-inflammatory properties. Nanoceria has been found toscavenge reactive oxygen species (ROS), possesssuperoxide-dismutase-like activity, prevent cardiovascular myopathy, andprovide radioprotection to normal cells from radiation as reported bythe following references: J. M. Perez et al., in Synthesis ofBiocompatible Dextran-coated Nanoceria with pH-dependent AntioxidantProperties. Small 2008, 4, 552-556; J. P. Chen et al., in Rare EarthNanoparticles Prevent Retinal Degeneration Induced by IntracellularPeroxides. Nature Nanotechnology 2006, 1, 142-150; J. Niu et al., inCardioprotective Effects of Cerium Oxide Nanoparticles in a TransgenicMurine Model of Cardiomyopathy. Cardiovasc Res 2007, 73, 549-559; R. W.Tarnuzzer et al., in Vacancy Engineered Ceria Nanostructures forProtection from Radiation-induced Cellular Damage. Nano Lett 2005, 5,2573-2577; and C. Korsvik et al., in Vacancy Engineered Ceria oxideNanoparticles Catalyze Superoxide Dismutase Activity, ChemicalCommunications 2007, 1056-1058.

The synthesis of biocompatible polymer-coated nanoceria with enhancedaqueous stability and unique pH-dependent antioxidant activity wasrecently reported by J. M. Perez et al. in Small 2008, 4, 552-556,supra. Particularly, it was found that nanoceria displays optimalantioxidant properties at physiological pH; whereas, it behaves as anoxidase at acidic pH, according to A. Asati et al., in Oxidase-LikeActivity of Polymer-Coated Cerium Oxide Nanoparticles, AngewandteChemie-International Edition 2009, 48, 2308-2312. Hence, this selectivebehavior may explain nanoceria's selective cytoprotection to normalcells, but not to cancer cells during radiation treatment or oxidativestress, as discussed by R. W. Tarnuzzer et al., in Nano Lett 2005, 5,2573-2577, supra.

In addition, the nature of the polymeric coating surrounding the ceriumoxide core could play a critical role in nanoceria's beneficial(antioxidant) vs harmful (oxidant) properties. It was hypothesized thatthe cytotoxicity of cerium oxide nanoparticles could depend upon theirsubcellular localization. Once inside the cells, the nanoceria particletoxicity could depend on whether the particles are localized inparticular cellular organelles, such as the lysosomes which are acidic,or distributed in the cytoplasm which is at neutral pH in normal cells.Since most tumors have an acidic microenvironment, this might switch offnanoceria antioxidant activity, turning on its oxidase activity andconsequently sensitizing the tumor towards radiation therapy.

There is always a need for another weapon in the arsenal needed to fightdisorders on a cellular level and the present invention provides theneeded weaponry.

In the prior art, naked, bare nanoceria particles have been reportedwherein the surface charge of the particle is modified. S. Patil et al.in “Protein Adsorption and Cellular Uptake of Cerium Oxide Nanoparticlesas a Function of Zeta Potential” Biomaterials, 2007 Nov., 28 (31):4600-4607 describes how surface chemistry of biomaterials have an impacton their performance. A. Vincent et al. in “Tuning Hydrated NanoceriaSurfaces: Experimental/Theoretical Investigations of Ion Exchange andImplications in Organic and Inorganic Interactions” Langmuir, 2010, 26(10), 7188-7198 teaches that surface charge modified hydrated ceriumoxide nanoparticles can be synthesized and provide detail of the dynamicion exchange interactions with the surrounding medium. These surfacecharge modifications were based on the use of naked, bare, uncoatednanoceria particles.

Other research by C. Wilhelm in “Intracellular Uptake of AnionicSuperparamagnetic Nanoparticles as a Function of Their Surface Coating”Biomaterials, 2003 24: 1001-1011 focused on the use of dextran-coatediron oxide nanoparticles to provide negative surface charges.

In co-pending U.S. patent application Ser. No. 12/704,678, with commoninventors and common ownership, it is reported that polymer-coatednanoceria has intrinsic oxidase activity at acidic pH values andnanoceria behaves as an oxidant at pH 4. It is also reported thatpolymer-coated cerium oxide nanoparticles bind to folate expressingcancer cells and can be detected via catalytic oxidation of sensitivecolorimetric substrates/dyes. The content of the co-pending U.S. patentapplication Ser. No. 12/704,678 is incorporated herein by reference.

In co-pending U.S. patent application Ser. No. 11/965,343, with commoninventors and common ownership, a method for synthesizing non-toxic,biodegradeable polymer coated nanoceria is disclosed. Thepolymeric-coatings discussed are at least one of a carbohydrate polymer,a synthetic polyol, a carboxylated polymer and derivatives thereof; thecontent of the co-pending U.S. patent application Ser. No. 11/965,343 isincorporated herein by reference.

A further co-pending U.S. patent application Ser. No. 12/169,179 withcommon inventors and common ownership, discloses polymer-coatednanoceria preparations that exhibit no toxicity to normal cells andexhibits pH-dependent antioxidant properties at neutral or physiologicalpH values and is inactive as an antioxidant at acidic pH values; the pHdependent properties of the polymer-coated nanoceria provides selectivecytoprotection. The content of co-pending U.S. patent application Ser.No. 12/169,179 is incorporated herein by reference.

SUMMARY OF THE INVENTION

A first objective of the present invention is to provide a surfacecoating on nanoceria to modulate its differential cytotoxicity behaviorin cancer versus normal cells.

A second objective of the present invention is to synthesize variousnanoceria preparations coated with either a polymer or a small molecule.

A third objective of the present invention is to synthesize variousnanoceria preparations coated with polyacrylic acid (PNC), aminatedpolyacrylic acid (ANC), or dextran (DNC) endowing the nanoparticles witha negative (−), positive (+) or neutral ( )) surface charge,respectively.

A fourth objective of the present invention is to provide fluorescentmodality to polymer-coated nanoceria, without compromising thesolubility of the nanoparticles in aqueous media or reducing the numberof available functional groups on the nanoparticle surface.

A fifth objective of the present invention is to encapsulate a dye, suchas DiI, so that the polymer-coated nanoparticles can be used forintracellular tracking of the nanoparticles.

A sixth objective of the present invention is to design a nanoparticlewherein the surface charge thereon is modulated to one of at least aneutral charge, a positive charge and a negative charge to determinecytotoxicity for specific cell lines.

The objectives set forth above are met by the present invention whichincludes a plurality of surface-coated cerium oxide nanoparticles with asurface charge on each particle wherein the surface charge is selectedfrom at least one of a positive charge, a negative charge and a neutralcharge exhibiting differential cellular internalization and subcellularlocalization in cells selected from normal, non-transformed cells andmalignant, transformed cells.

It is preferred that the plurality of surface-coated cerium oxidenanoparticles with a positive (+) surface charge are coated withaminated polyacrylic acid (ANC). It is also preferred that the pluralityof surface-coated cerium oxide nanoparticles with a negative (−) surfacecharge are coated with polyacrylic acid (PNC). Another preferred surfacecoating for the plurality of cerium oxide nanoparticles provides aneutral (0) surface charge when coated with dextran (DNC) polymer.

It is also preferred that the plurality of polymer-coated cerium oxidenanoparticles further include an encapsulating dye within thehydrophobic microdomains of the polymeric coating on the surface of eachnanoceria particle thereby fluorescently labeling the nanoparticles.

In the present invention, normal, non-transformed cells are isolated andselected from the group consisting of cardiac myocytes (H9c2) and humanembryonic kidney cells (HEK293) and malignant, transformed cells areisolated and selected from the group consisting of lung carcinoma cells(A549) and breast carcinoma cells (MCF-7).

It is preferred that nanoceria particles with a positive (+) surfacecharge are internalized in isolated cells, selected from at least one ofnormal cardiac myocytes (H9c2), normal human embryonic kidney cells(HEK293) and malignant lung cells (A549). It is also preferred thatpolymer coated nanoparticles, with a positive surface charge areinternalized in the normal cardiac myocytes (H9c2) and subsequentlylocalized in the lysosome of each cell where the nanoparticle oxidaseactivity exhibits cytotoxicity.

In the present invention, polymer-coated cerium oxide nanoparticles,with a positive surface charge are internalized in normal humanembryonic kidney cells (HEK293) and subsequently localized in thelysosome of each cell wherein the nanoparticle oxidase activity exhibitscytotoxicity. The polymer-coated cerium oxide nanoparticles, with apositive surface charge are also internalized in malignant lung cells(A549) and subsequently localized in the lysosome of each cell where thenanoparticle oxidase activity exhibits cytotoxicity.

It was also determined that the polymer-coated nanoparticles, with apositive surface charge are internalized in malignant breast tumor cells(MCF-7) and do not localize to the lysosome of each cell resulting in nocytotoxicity.

With regard to the plurality of surface-coated cerium oxidenanoparticles with a negative (−) surface charge, cell internalizationoccurred in normal cardiac myocytes (H9c2) and normal human embryonickidney cells (HEK293) and subsequent subcellular localization alsooccurred in the cytoplasm of said normal cells and no cytotoxicity wasdisplayed. It was also determined that the nanoparticle with a negative(−) surface charge undergoes cell internalization in malignant lungcarcinoma cells (A549) and subcellular localization into the lysosomesof lung carcinoma cells (A549) and displays cytotoxicity.

A further determination was made that the nanoparticle with a negative(−) surface charge undergoes no cellular internalization in malignantbreast carcinoma cells (MCF-7) and no subcelluar localization inmalignant breast carcinoma cells (MCF-7) and displays no cytotoxicity.

Research shows that the plurality of surface-coated cerium oxidenanoparticles with a neutral (0) surface charge undergoes cellinternalization in normal, non-transformed cells and malignant,transformed cells and subcellular localization in the cytoplasm of saidcells and exhibits no toxicity.

A preferred pharmaceutical composition of the present invention includesa plurality of surface-coated cerium oxide nanoparticles and a carrierfor selective cytotoxicity of malignant lung tumor cells.

Another preferred composition includes at least one cerium oxidenanoparticle; and a polymer coating bound to said cerium oxidenanoparticle, wherein said polymer coating provides a surface charge tosaid composition. It is preferred that the polymer coating is selectedfrom at least one of polyacrylic acid that provides a negative surfacecharge to said composition, aminated polyacrylic acid that provides apositive surface charge, and dextran that provides a neutral surfacecharge.

The preferred composition further includes a fluorescent modality whichis a dye encapsulated by said polymer coating; the preferred dye is1,1′-dioctadecyl-3,3,3′3′-tetramethylindocarbocyanine perchlorate (DiI).

A preferred method for localizing at least one nanoparticle into atleast one lysosome of at least one tumor cell includes providing atleast one cerium oxide nanoparticle coated with a polymer coating,wherein said polymer coating provides a negative surface charge;exposing said cerium oxide nanoparticle to the tumor cell and contactingsaid tumor cell with the polymer-coated cerium oxide nanoparticle sothat the nanoparticle is localized in the lysosome of the tumor cellproviding a cytotoxic result.

Another preferred composition for intracellular localization ofnanoparticles includes at least one cerium oxide nanoparticle, and atleast one polymer coating bound to said nanoparticle, wherein saidcoating imparts a specific surface charge, said surface charge beingdependant on a desired intracellular location. It is preferred that thepolymer coating include polyacrylic acid, and said specific surfacecharge is negative, and said desired intracellular location is at leastone lysosome.

It is also preferred that the polymer coating is dextran, and saidsurface charge is neutral, and the desired intracellular location is thecytoplasm. The preferred composition further includes a fluorescentmodality comprising a dye encapsulated by said polymer coating whereinsaid dye is DiI.

A preferred method for intracellular localization of at least onenanoparticle includes providing at least one cerium oxide nanoparticlewith a polymer coating, wherein said polymer coating has a specificsurface charge, said charge depending on a desired intracellularlocation, and exposing the polymer coated cerium oxide nanoparticles tocells. When the polymer coating is polyacrylic acid, the specificsurface charge is negative, and the desired intracellular location is atleast one lysosome. It is also preferred that the polymer coating isdextran, said surface charge is neutral, and desired intracellularlocation is the cytoplasm.

Another preferred method for non-cytotoxic delivery of particles tocells includes providing at least one cerium oxide nanoparticle, whereinat least one polymer coating is bound to said cerium oxide nanoparticle,wherein said polymer coating provides a surface charge that is at leastone of a positive charge and a neutral charge, and exposing thepolymer-coated cerium oxide nanoparticle to at least one cell. A furtherpolymer coating comprises dextran that provides the neutral charge; andanother polymer coating includes aminated polyacrylic acid that providesa positive charge.

A preferred method for killing at least one tumor cell includesproviding at least one cerium oxide nanoparticle coated with a polymercoating, wherein said polymer coating provides a negative surfacecharge, and exposing the cerium oxide nanoparticle to the tumor cell.

A preferred kit for treating cancer includes a quantity of cerium oxidenanoparticles coated with a polymer providing a surface charge, and atleast one medical application device, wherein said medical applicationdevice and said nanoparticles are packaged together. It is preferredthat the medical application device is selected from at least onesyringe one topical applicator and one pill.

A preferred surface coated nanoceria particle with a neutral surfacecharge that is administered to a plurality of isolated normal and aplurality of isolated transformed cells and cellular uptake does notoccur resulting in no toxicity to the normal and transformed cells.

Another preferred surface coated nanoceria particle with a neutralsurface charge that is administered to a plurality of isolated normaland a plurality of isolated transformed cells and cellular uptake doesoccur with subsequent subcellular localization in the cytoplasm of eachnormal and transformed cell resulting in no toxicity to the normal andtransformed cells.

Further objects and advantages of this invention will be apparent fromthe following detailed description of a presently preferred embodiment,which is illustrated in the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows surface functionalization of cerium oxide nanoparticleswherein cerium oxide nanoparticles with different polymer coatings andsurface modifications yield nanoparticles with a negative [PNC(−)], apositive [ANC(+)], and a neutral [DNC(0)] charge. A fluorescent dye(DiI), 100, 110, 120 was encapsulated using a modified solvent diffusionmethod.

FIG. 2A is a chart of the size of a cerium oxide nanoparticle coremeasured by transmission electron microscopy (TEM).

FIG. 2B is a graph of the Zeta potential of cerium oxide nanoparticleswith different surface functionalities: negative, neutral and positive.

FIG. 2C is the FT-IR spectra of the carboxylated (negative) and aminated(positive) surface group on nanoceria.

FIG. 2D is the fluorescence emission spectra of the DiI-encapsulatingnanoceria and free dye DiI.

FIG. 3A is a black and white line drawing of a confocal image showinguptake of PNC (−) coated cerium oxide nanoparticles (nanoceria) bynormal H9c2 cardiac myocyte cells; minimal uptake is observed.

FIG. 3B is a black and white line drawing of a confocal image showinguptake of ANC (+) coated cerium oxide nanoparticles (nanoceria) bynormal H9c2 cardiac myocyte cells; uptake observed is shown by irregularshapes.

FIG. 3C is a black and white line drawing of a confocal image showinguptake of DNC (0) coated cerium oxide nanoparticles (nanoceria) bynormal H9c2 cardiac myocyte cells; diffused cytoplasmic localizationwith internalization in H9c2 is shown by interconnected irregularshapes.

FIG. 4A is a black and white line drawing of a confocal image of A549lung carcinoma cells after treatment with PNC(−) coated nanoceria for 3hours (3 h); PNC (−) coated nanoceria particles are uptaken by A549 lungcarcinoma cells at a minimal level.

FIG. 4B is a black and white line drawing of a confocal image of A549lung carcinoma cells after treatment with ANC(+) for 3 hours (3 h);positively charged particles are taken up by A549 lung carcinoma celllines to a greater degree than the negatively charged or neutral chargecoated nanoparticles.

FIG. 4C is a black and white line drawing of a confocal image of A549lung carcinoma cells after treatment with DNC(0) coated nanoceria for 3h; DNC(0) coated nanoceria is internalized in the A549 cells, in amanner suggesting that these coated nanoparticles are localized in thecytosol with a small fraction confined within the endosomalcompartments.

FIG. 5A is a black and white line drawing of a confocal image of ANC(+)coated nanoceria intracellular localization in lung carcinoma cells(A549); on internalization, the positively charged [ANC (+)] nanoceriaco-localizes with the lysosome.

FIG. 5B is a black and white line drawing of a confocal image of PNC(−)coated nanoceria intracellular localization in lung carcinoma cells(A549); on internalization, the negatively charged [(PNC (−)] nanoceriaco-localizes with the lysosome.

FIG. 5C is a black and white line drawing of a confocal image of DNC(0)coated nanoceria intracellular localization in lung carcinoma cells(A549); on internalization, the neutral nanoceria [DNC (0)] localizesmostly in the cytoplasm.

FIG. 6A is a black and white line drawing of a confocal image of ANC(+), coated nanoceria intracellular localization in cardiac myocytes(H9c2); on internalization, the posivitely charged [ANC (+)] is found inboth the cytoplasm and lysosomes.

FIG. 6B is a black and white line drawing of a confocal image of DNC(0)coated nanoceria intracellular localization in cardiac myocytes (H9c2);on internalization, the neutral nanoceria [DNC (0)] does not co-localizesignificantly to the lysosomes with most of the nanoparticles localizingto the cytoplasm.

FIG. 7A is a graph of lysosomal isolation and determination of theoxidase-like activity of ANC(+), PNC(−) and DNC(0) coated nanoceria inH9c2 cardiac myocyte cell lines; the ANC (+) coated nanoparticles aremostly entrapped into lysosomes, based on the presence of significantoxidase activity in the lysosomes isolated from these cells lines afterincubation with ANC (+).

FIG. 7B is a graph of ANC(+), PNC(−) and DNC(0) coated lysosomalisolation and determination of the oxidase-like activity of nanoceria inHEK293 human embryonic kidney cell lines; the ANC (+) coatednanoparticles are mostly entrapped into lysosomes, based on the presenceof significant oxidase activity in the lysosomes isolated from thesecells lines after incubation with ANC (+).

FIG. 7C is a graph of lysosomal isolation and determination of theoxidase-like activity of ANC(+), PNC(−) and DNC(0) coated nanoceria inA549 lung carcinoma cell lines; the ANC (+) and PNC(−) coatednanoparticles are mostly entrapped into lysosomes, based on the presenceof significant oxidase activity in the lysosomes isolated from thesecells lines after incubation with ANC (+) and PNC(−) coated nanoceria.

FIG. 7D is a graph of lysosomal isolation and determination of theoxidase-like activity of ANC(+), PNC(−) and DNC(0) coated nanoceria inMCF-7 breast carcinoma cell lines; the ANC (+), PNC(−) and DNC(0) coatednanoparticles are not internalized or not entrapped into lysosomes. Inthe absence of significant oxidase activity in the lysosomes isolatedfrom these MCF-7 breast cancer cells lines after incubation with ANC(+), PNC(−) and DNC(0) coated nanoceria, there was no toxicity to theMCF-7 breast cancer cell lines.

FIG. 8A is a graph showing cytotoxicity of ANC(+), PNC(−) and DNC(0)coated cerium oxide nanoparticles to H9c2 cardiac myocyte cells.

FIG. 8B is a graph showing cytotoxicity of ANC(+), PNC(−) and DNC(0)coated cerium oxide nanoparticles to HEK293 human embryonic kidneycells.

FIG. 8C is a graph showing cytotoxicity of ANC(+), PNC(−) and DNC(0)coated cerium oxide nanoparticles to A549 lung carcinoma cells.

FIG. 8D is a graph showing cytotoxicity of ANC(+), PNC(−) and DNC(0)coated cerium oxide nanoparticles to MCF-7 breast carcinoma cells.

FIG. 9A is a graph showing cytotoxicity of ANC(+) and PNC(−) coatedcerium oxide nanoparticles on H9c2 cardiac myocytes in the presence ofan endocytosis inhibitor; no toxicity was measured.

FIG. 9B is a graph showing cytotoxicity of ANC(+), PNC(−) and DNC(0)coated cerium oxide nanoparticles on A549 lung carcinoma cells in thepresence of an endocytosis inhibitor; no significant toxicity measured.

FIG. 10A is a graph showing cytotoxicity of negatively charged aminatedpolymer coated iron oxide nanoparticles [IONP(−)] and positively chargedpolymer coated iron oxide nanoparticles[IONP(+)] on A549 lung carcinomacells.

FIG. 10B is a graph showing cytotoxicity of negatively charged aminatedpolymer coated iron oxide nanoparticles [IONP(−)]and positively chargedaminated polymer coated iron oxide nanoparticles [IONP(+)] on H9c2cardiac myocyte cells.

FIG. 11 is a schematic representation of polymer-coated nanoceria withdifferent surface charges displaying cell internalization, discretesubcellular localization and toxicity profiles. Neutral DNC(0) coatednanoceria internalizes and localizes mostly into the cytoplasm 130 ofcells and hence it is not cytotoxic. Whereas, ANC(+) and PNC(−) canlocalize either into the cytoplasm 140 or the lysosomes 170, dependingon the type of cells. When the nanoceria localizes to the lysosome 160,170, 180, the low pH of this organelle activates the nanoparticle'soxidase-like activity, exhibiting toxicity. ANC(+) or PNC(−) thatlocalizes into the cytoplasm 150 displays no cytotoxicity.

DESCRIPTION OF THE PREFERRED EMBODIMENT

Before explaining the disclosed embodiment of the present invention indetail, it is to be understood that the invention is not limited in itsapplication to the details of the particular arrangement shown since theinvention is capable of other embodiments. Also, the terminology usedherein is for the purpose of description and not of limitation.

It would be useful to discuss the meanings of some words and acronymsused herein and their application before discussing the composition ofmatter and method of using and making the same.

A549 is used to identify lung carcinoma cells

ANC(+) is used herein to mean an aminated polymer-coated nanoceriaparticle with a positive charge on the surface of the particle.

DiI is a fluorescent dye with the chemical name,1,1′-dioctadecyl-3,3,3′3′-tetramethylindocarbocyanine perchlorate.

DNC(0) is used herein to mean a dextran polymer-coated nanoceriaparticle with a neutral charge on the surface of the particle.

H9c2 is used to identify cardiac myocyte cells

HEK293 stands for human embryonic kidney cells

IONP(+) is used herein to mean a polymer coated iron oxide nanoparticlewith a positive charge on the surface of the particle.

IONP(−)] is used herein to mean an aminated polymer coated iron oxidenanoparticle with a negative charge on the surface of the particle.

MCF-7 is used to identify breast carcinoma cells

PNC(−) is used herein to mean a polyacrylic acid coated nanoceriaparticle with a negative charge on the surface of the particle.

Surface coatings on nanoceria particles are disclosed herein aspolymeric coatings; however, the coatings can comprise small molecules,such as amino acids, small cyclical polyols such as cyclodextrins, andsimple sugars. Thus, the examples and discussion herein based onpolymeric-coatings should not be considered as limiting. The criteriafor a suitable coating for the present invention is one that adheres tothe surface of the nanoceria particle, provides a surface charge for thenanoceria particle and at the same time modulates the cytotoxicity ofthe resulting coated nanoceria nanoparticle.

In the present invention, a novel use of a polymer'ssurface-charge-dependent cell internalization and cytotoxicity profileof cerium oxide nanoparticles in normal versus malignant cells isreported. Various cell lines were selected in order to assess thecorresponding behavior of cerium oxide nanoparticles. Cardiac myocytes(H9c2) and human embryonic kidney (HEK293) cells were selected asnon-transformed (normal) cells, whereas lung (A549) and breast (MCF-7)carcinomas were selected as transformed (cancer) cell lines.

Except for breast carcinoma cells, results show that positively chargednanoceria particles internalize in other cells, localize preferentiallyin lysosomes and subsequently become toxic to these cells. With regardto polymer-coated nanoceria with a negative charge, it was internalizedand localized into the lysosomes of lung carcinoma (A549) cells only,but not by the lysosomes of breast carcinoma cells (MCF-7), thusexhibiting toxicity only to the lung carcinoma cells.

Also, the negatively charged polymer-coated nanoceria particles were notinternalized and therefore were not toxic to the normal cells,including, but not limited to, cardiac myocytes and human embryonickidney cells. Surprisingly, nanoceria with a neutral charge was nottoxic to normal cells or cancer cells, as these nanoparticles primarilylocalized in the cytoplasm of these cells. The combined results suggestthat the internalization and subcellular localization of polymer-coatednanoceria plays a critical role in the toxicity profile of thisnanomaterial. The results herein also suggest that the coating onnanoceria can be engineered in order to modulate its differentialcytotoxicity behavior in cancer versus normal cells.

EXAMPLE 1 Synthesis and Characterization of Polymer-Coated Cerium OxideNanoparticles

FIG. 1 is an illustration of various nanoceria preparations coated witheither polyacrylic acid (PNC), aminated polyacrylic acid (ANC), ordextran (DNC) that endow nanoparticles with a negative (−), positive (+)or neutral (0) surface charge, respectively.

During synthesis, the nanoparticles were labeled fluorescently, byencapsulating a dye (DiI) 100, 110, 120 within the hydrophobicmicrodomains of the polymeric coatings of each nanoceria preparation,following a previously reported solvent diffusion methodology disclosedby S. Santra in Drug/dye-loaded, Multifunctional Iron oxideNanoparticles for Combined Targeted Cancer Therapy and DualOptical/Magnetic Resonance Imaging, Small 2009, 5, 1862-1868.

Transmission electron microscopy (TEM) studies reported in FIG. 2A showthe presence of nanoparticles of similar core size (3 to 4 nm) in allpreparations as reported by A. Asati, et al., in AngewandteChemie-International Edition 2009, 48, 2308-2312, supra. While dynamiclight scattering experiments, show the presence of monodispersenanoceria preparations with average hydrodynamic diameter of 14 nm forDNC(0) and 5 nm for both PNC (+) and ANC(+).

The presence of different surface charges in the various nanoceriapreparations was assessed by zeta potential as shown in FIG. 2B,confirming the presence of a negative, neutral and positive charge forPNC, DNC and ANC, respectively. FT-IR analysis further confirmed thenanoparticle polymer surface coating and functionality in FIG. 2C.

When using the methodology reported by S. Santra in Small 2009, 5,1862-1868 supra, fluorescent modality was introduced to thepolymer-coated nanoceria, without compromising the solubility of thenanoparticles in aqueous media or reducing the number of availablefunctional groups on the nanoparticle surface. Successful encapsulationof DiI into the nanoceria was confirmed via fluorescence spectroscopy,where a blue shift in the DiI-encapsulating ceria nanoparticles (584 nm)emission spectrum was observed as opposed to free DiI (592 nm) as shownin FIG. 2D. The DiI-encapsulating polymer-coated-nanoceria displayedgood aqueous stability over long periods of time without significantrelease of the dye, and can be used for the intracellular tracking ofthe nanoparticles.

Thus, surface functionalization of cerium oxide nanoparticles as shownin FIG. 1 occurs during the synthesis of the polymer-coatednanoparticles via a solvent diffusion method.

EXAMPLE 3 Surface-Charge-Dependent Cellular Interaction of Nanoceria

Confocal microscopy experiments were performed in order to study thecellular uptake and intracellular localizaton of the DiI-labeledpolymer-coated nanoceria. In these experiments, PNC (−), ANC (+) and DNC(0) (1.0 mM) were incubated with two transformed carcinoma cell lines(A549 lung and MCF-7 breast carcinomas), and two non-transformed(normal) cell lines (H9c2 cardiac myocytes and HEK293 human embryonickidney cells). These cells lines were selected in order to investigatewhether there is a difference in uptake by ANC(+), PNC(−) and DNC(0)polymer-coated nanoparticles, thus potentially displaying differenttoxicity profiles.

First, the internalization pattern of ANC(+), PNC(−) and DNC(0) wasstudied in normal cardiac myocyte cells (H9c2), using 10,000 cells asshown in FIGS. 3A-3C. After a 3-hour incubation, results showed that thenegatively charged carboxylated nanoparticles [PNC(−)] were minimallyuptaken by the H9c2 cardiac myocyte cells and did not internalize in theH9c2 cell line as shown in FIG. 3A.

The positively charged aminated nanoparticles [ANC(+)] were uptaken bythe H9c2 cardiac myocytes, as indicated by the elevated cell-associatedfluorescence in the H9c2 cells as shown by irregular shapes in FIG. 3B.

The neutral dextran-coated nanoparticle [DNC(0)] shows a higher degreeof internalization in the H9c2 cells as shown in FIG. 3C. Interestingly,the DNC(0) intracellular fluorescence pattern in the H9c2 cells wasdiffused, showing a different pattern from the ANC(+) punctuatedfluorescence pattern. This difference seems to indicate a uniquenanoparticle surface-charge dependent internalization mechanism withdifferent intracellular compartmentalization into the cytoplasm amongthe nanoparticles studied in the non-transformed cell line.

EXAMPLE 4 Internalization Experiments with Cancer Cells

FIGS. 4A-4C show results of internalization experiments performed withcancer cells (10,000 cells) derived from lung (A549) carcinoma cells.PNC(−) uptake by the A549 cells is minimal or slight as shown in FIG.4A. In FIG. 4B, lung (A549) carcinoma cells were able to uptake ANC (+);interestingly, a higher degree of internalization and pronouncedpunctuated fluorescence was observed with the ANC(+) in the A549 cells.This might indicate that in these cells the majority of the ANC(+)localized into endosomal compartments. In contrast, in FIG. 4C, minimaland diffused intracellular fluorescence was observed with the DNC (0) inthe A549 cells, suggesting that these nanoparticles are mostly localizedin the cytosol with a small fraction confined within the endosomalcompartments. Taken together, the above results suggest that the surfacecharge on polymer-coated nanoceria dictates their differentialinternalization and localization in normal cells (FIGS. 3A-3C) versuscancer cells (FIGS. 4A-4C).

EXAMPLE 5 Intracellular Distribution of Polymeric Cerium OxideNanoparticles

To corroborate that after internalization some of the polymer-coatednanoceria localized in endosomal compartments, we first treated thecells (10,000 cells) for 3 h with the PNC(−), ANC(+) and DNC(0) (1.0 mM)followed by a 20-minute treatment with Lysotracker (35 nM), a lysosomespecific dye. Lysotracker is a green fluorescent dye that stains theacidic lysosomes, hence the potential co-localization betweenDiI-labeled-nanoceria (red) and lysosomes (green) should yield ayellow/orange overlap when the images are merged.

Experiments were carried out with A549 lung carcinoma cells. Resultsshowed that DiI-labeled-ANC(+) and DiI-labeled-PNC(−) co-localized withLysotracker in the A549 lung cancer cells, as determined by confocalmicroscopy images shown in FIGS. 5A-5B. Even though less internalizationof DiI-labeled-PNC(−) is observed in these cells, the internalizednanoparticles predominantely co-localized mostly with the Lysotrackerdye indicating lysosomal localization.

In contrast, DiI-labeled-DNC(0) showed a diffused distribution withminimal localization in the lysosomes of A549 cells as shown in FIG. 5C.The results in Example 5 demonstrate that the surface charge on theceria nanoparticles dictates the subcellular localization of nanoceriain cancer cells.

EXAMPLE 6 Determination of the Oxidase-Like Activity ofLysosome-Residing Nanoceria

Nanoceria has been reported by A. Asati et al., in AngewandteChemie-International Edition 2009, 48, 2308-2312, supra to possessunique oxidase-like activity at acidic pH, oxidizing variouscolorimetric substrates, such as 3,3′,5.5′-tetramethylbenzydine (TMB)and 2,2-azino-bis(3-ethylbenzothizoline-6-sulfonic acid (AzBTS). Thisactivity can be employed to assess the localization of nanoceria invarious cell organelles, particularly lysosomes, via the nanoparticle'soxidase activity, using TMB as the colorimetric substrate.

Hence, to further confirm that some of the polymer-coated cerium oxidenanoparticles localized into the lysosomes, cells were incubated withthe different nanoceria preparations for 3 hours, followed by lysosomalisolation. Then, the oxidase activity of the isolated lysosomes wasdetermined spectrophotometrically via TMB oxidation. As expected and inagreement with the microscopy and lysosomal co-localization experiments,we found that ANC(+) coated nanoceria particles were mostly entrappedwithin the lysosome of the H9c2 cardiac myocyte cell lines, as thelysosomes isolated from these cells exhibited significant levels ofoxidase activity as shown in FIG. 6A.

On internalization, the positively charged ANC(+) coated nanoceria isfound in both the cytoplasm and lysosomes. In contrast, neutralnanoceria with DNC(0) coating does not significantly co-localize to thelysosomes with most of the nanoparticles found in the cytoplasm asobserved in FIG. 6B.

Referring now to FIGS. 7A-7D, lysosomal isolation and determination ofoxidase-like activity of entrapped polymer-coated nanoceria is reported.In agreement with the microscopy and lysosmal co-localizationexperiments, it was determined that ANC(+) coated nanoceria were mostlyentrapped within the lysosome of the H9c2, HEK293 and A549 cell lines,as lysosomes isolated from these cells exhibited significant levels ofoxidase activity as shown in FIGS. 7A-7C.

In MCF-7 breast carcinoma cell line, no localization to the lysosomeswas observed as these cells do no uptake any of the nanoparticles asshown in FIG. 7D.

Interestingly, when cells were incubated with PNC(−), only the lysosomesisolated from A549 lung carcinoma cells (FIG. 7C) exhibited oxidaseactivity based on the lysosomal co-localization experiments. Meanwhile,the lysosomes from cells incubated with the DNC(0) exhibit minimaloxidase activity, corroborating the confocal microscopy studies.Lysosomes isolated from untreated cells (H9c2, HEK293, A549 and MCF-7)did not possess any oxidase activity (control), as indicated by theabsence of TMB oxidation shown in FIGS. 7A-7D, and confirming theabsence of endogenous oxidase activity in these organelles.

In H9c2 and HEK293 cell lines, the ANC (+) nanoparticles are mostlylocalized into lysosomes, judging by the presence of significant oxidaseactivity in the lysosomes isolated from these cells lines afterincubation with ANC (+) as shown in FIG. 7A and FIG. 7B, respectively.PNC (−) and DNC (0) treated cells showed minimal oxidase activity intheir lysosomes as shown in FIGS. 7A, 7B and 7D. In A549, oxidaseactivity was detected in cells treated with ANC (+) and PNC(−), whileminimal activity was present in the DNC(0) treated cells (FIG. 7C).

The lysosomes isolated from MCF-7 cells treated cells did not showsignificant oxidase activity as the polymer-coated nanoceria does notinternalized in these cell lines (FIG. 7D). Lysosomes isolated fromnon-treated cells do not show any oxidase activity.

EXAMPLE 8 Intracellular-Distribution-Dependent Cytotoxicity of PolymericCerium Oxide Nanoparticles

In order to determine if the surface-charge-dependent internalizationand intracellular (lysosomal vs cytoplasmic) localization of thepolymer-coated nanoceria plays a role in the nanoparticle cytotoxicity,cell viability (MTT) assays were performed. Interestingly, we found thatDNC(0) did not exhibit any toxicity to the cell lines studied (FIGS.8A-8D), as most of these nanoparticles localized to the cytoplasm.Furthermore, prolonged incubation of these cell lines with thedextran-coated-nanoceria did not affect cell morphology andproliferation ability as previously reported by J. M. Perez et al., inSmall 2008, 4, 552 -556, supra.

In contrast, ANC(+) and PNC(−) had different degrees of toxicity,depending on the nanoparticle's localization inside the cell. Forinstance, the PNC(−) nanoparticles were not toxic to the H9c2 cardiacmyocytes or the HEK293 kidney cells as shown in FIGS. 8A and 8B,respectively. PNC(−) coated nanoceria particles were toxic to the A549lung carcinoma cells (FIG. 8C). This can be explained by the minimaluptake of PNC(−) by normal cells, as opposed to the A549 lung cancercells that exhibited enhanced nanoparticle uptake and localization tothe lysosomes (FIG. 7C). ANC (+) also exhibited various degrees oftoxicity. Notably, the ANC(+) polymer-coated nanoparticles were moretoxic to H9c2 (FIG. 8A), HEK293 (FIG. 8B), and A549 (FIG. 8C) cells,since these cells exhibited increased nanoparticle internalization andlysosomal localization.

In FIG. 8D, the breast cancer cells (MCF-7) did not show any toxicityafter treatment with any of the polymer-coated nanoparticles studied,since these cells did not show any significant nanoparticle uptake.

In FIGS. 8A-8D, MTT assays show that the ANC (+) is cytotoxic to allcell lines except for the breast carcinoma cells. While PNC (−) is onlycytotoxic to A549 lung cancer cells as they internalize and localizedinto the lysosomes these cells. DNC (0) does not show any toxicity toany of the cell lines.

Incubation of the H9c2 cardiac myocytes (FIG. 9A) and A549 lungcarcinoma cells (FIG. 9B) with ANC(+) and PNC(−) in the presence ofinhibitors of endocytic pathway, such as, 2-deoxyglucose and sodiumazide, abrogates the cytotoxicity of the nanoparticle. FIGS. 9A and 9Bconfirm that an endocytic uptake of these polymer-coated nanoparticlesand eventual localization to lysosomes was responsible for theircellular toxicity. Taken together, these results demonstrate thatlocalization of nanoceria into lysosomes (an acidic cell compartment),as opposed to localization into the cytoplasm, leads to cytotoxicity byactivating the oxidase activity of nanoceria within these organelles.

EXAMPLE 9 Comparison of the Toxicity of Surface-Charge-EngineeredNanoceria and Iron Oxide Nanoparticles

To demonstrate that the observed cytotoxicity of nanoceria is attributedto the cerium oxide core (oxidase activity) and not to the nature of thepolymeric coating, we performed experiments with iron oxide particles.Specifically, DiI-labeled-aminated polyacrylic acid [IONP(+)] with apositive surface charge and DiI-labeled carboxylated polyacrylic acid[IONP(−)] with a negative surface charge were used to coat iron oxidenanoparticles.

Polymer-coated iron oxide nanoparticles have been widely used in variousapplications, particularly in Magnetic Resonance Imaging (MRI) withminimal toxicity. For instance, various preparations of dextran-coatediron oxide nanoparticles are used in the clinic for liver and lymph nodemetastasis imaging, as discussed by R. Weissleder in Liver MR Imagingwith Iron Oxides: Toward Consensus and Clinical Practice. Radiology1994, 193, 593-595; M. G. Harisinghani et al., in Noninvasive Detectionof Clinically Occult Lymph-Node Metastases in Prostate Cancer, N Engl JMed 2003,348, 2491-2499; and M. G. Harisinghani et al., in DoesContrast-enhanced Imaging Alone Suffice for Accurate Lymph NodeCharacterization?, AJR Am J Roentgenol 2006, 186, 144-148.

The polymeric iron oxide nanoparticles [IONP(+)and IONP(−)] were nottoxic to either the transformed A549 carcinoma cell line or thenon-transformed H9c2 cell line as shown in FIGS. 10A and 10B,respectively. The fact that more of the IONP(+) nanoparticles werelocalized into the lysosomes upon internalization in H9c2 did not seemto significantly alter the toxicity profile of the polymer coated ironoxide nanoparticles. These results are not surprising, as iron oxidenanoparticles do not possess oxidase activity, particularly at the lowpH of the lysosomes. Therefore, we can conclude that the intrinsicoxidase behavior of cerium oxide nanoparticles is responsible for thenanoparticle's cytotoxicity, particularly when they localize into acidiccell compartments such as lysosomes. Cytotoxicity experiments with theneutral dextran-coated iron oxide nanoparticles were not performed sinceit is well established that these nanoparticles are non-toxic.

In FIG. 11, the schematic drawing shows polymer-coated nanoceria withdifferent surface charges displaying cell internalization, discretesubcellular localization and toxicity profiles. Neutral DNC(0)internalizes and localizes mostly into the cytoplasm 130 of cells andhence it is not cytotoxic. Whereas, ANC(+) and PNC(−) can localizeeither into the cytoplasm 140 or the lysosomes 170, depending on thetype of cells. When ANC(+) or PNC(−) localize into the cytoplasm 150 nocytotoxicity is displayed. Thus, no toxicity was observed whenpolymer-coated nanoceria localized to the cytoplasm 130, 140, 150 ofcells, however, upon localization to the lysosomes 160, 170, 180 the lowpH of this organelle activates the oxidase-like activity of the ceriananoparticle, exhibiting toxicity.

Materials and Methods Synthesis of Polymer-Coated Nanoceria Preparations[DNC (0), PNC (−) and ANC (+)]

The polymer coated nanoceria preparations, DNC (0) and PNC (−) weresynthesized using the methodology described by A. Asati et al., inAngewandte Chemie-International Edition 2009, 48, 2308-2312 supra.Briefly, a solution of cerium (III) nitrate (2.17 g, 1.0 M, Aldrich,99%) in water (5.0 mL) was mixed separately with an aqueous solution ofeither polyacrylic acid (PAA, 0.5 M, Sigma) or dextran (1.0 M, Sigma)and mixed it thoroughly using a vortex mixer. The resulting mixture wasthen added to an ammonium hydroxide solution (30.0 mL, 30%, SigmaAldrich) under continuous stirring for 24-h at room temperature.

The preparation was then centrifuged at 4000 rpm for two 30-minutecycles to settle down any debris and large agglomerates. The supernatantsolution was then purified from free polymers and other reagents andthen concentrated using a 30K Amicon cell (Millipore Inc.).ANC (+) issynthesized directly from PNC (−). In this method, PNC (−) (5.0 mL, 1.5mg/mL) is treated with EDC[1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide]solution (28.0 mg, 30mmol) inMES buffer (500 μL, 0.1 M, pH 6.0) followed by the drop-wiseaddition of N-Hydroxy succinamide (NHS) solution (22.0 mg, 30 mmol)inMES buffer (500 μL, 0.1 M, pH 6.0) and incubated for 3 minutes at roomtemperature. Ethylenediamine (EDA, 10 mg, 25 mmol) in DMSO (100 μL) isthen added drop-wise to the final reaction mixture and stirred for anadditional 3 h at room temperature.

The resulting solution was purified to remove excess EDA and otherreagents usingan Amicon dialysis membrane (MWCO 30K) from Millipore. Thefinal ANC (+) preparation (1.5 mg/mL) in DI water was stored in therefrigerator for further characterization.

DiI Encapsulation in Polymeric-Coated Nanoceria [DNC (0), PNC (−) andANC (+)]

In order to encapsulate the DiI dye (Invitrogen) into the polymericmatrix of the various polymer-coated nanoceria, we have used a modifiedsolvent diffusion method, as reported by S. Santra in Small 2009, 5,1862-1868 supra. Briefly, to a 4.0 mL of the nanoparticle solution [1.5mg/mL, DNC (0), PNC (−) or, ANC (+)], 200.0 μL of DiI solution [6.0 μLof DiI (10 mg/mL) in 194 μL of DMSO] was added dropwise while mixing(1000 rpm) at room temperature. Afterwards, the preparation was dialyzed(MWCO 30 K) against deionized water to remove any free DiI, and finallywas dialyzed overnight in phosphate-buffered saline (1× PBS) toreconstitute the final preparation in PBS.

Characterization

The various polymer-coated nanoceria preparations were charecterized bytransmission electron microscopy (TEM) to confirm the size of thenanocrystal core. TEM was performed by mounting a drop of nanoparticleson a holey carbon-coated copper 400-mesh grid (2SPI, USA) and imageswere taken on a FEI TECNAI F30 microscope operating at 300 kV.Surfacecharge on the nanoparticle was confirmed by the zeta potentialmeasurements (Malvern Zetasizer and disposable zeta cells). FT-IRexperiments were performed on vacuum dried samples to verify the surfacefunctionalities on nanoparticles (Perkin Elmer Spectrum 100 FT-IRspectrometer). Fluorescence spectroscopy studies were done onDiI-labeled using a Nanolog HORIBA JOBIN YVON Spectrometer to confirmthe DiI dye encapsulation in nanoparticles.

Cell Culture

All cell lines in this study [lung carcinoma (A549), cardiac myocytes(H9c2), human embryonic kidney (HEK293) and breast carcinoma (MCF-7)]were obtained from American Type Culture Collection (ATCC) in Manassas,Va. The cardiomyocytes were grown in Eagle's Minimal Essential mediumsupplemented with fetal bovine serum (10%), sodium pyruvate,L-glutamine, penicillin, streptomycin, amphotericin B and non-essentialamino acids. HEK293 cells were grown in Eagle's Minimal Essential mediumsupplemented with fetal bovine serum (10%) and 1% penicillin. The lungcancer cells were grown in Kaighn's modification of Ham's F12 medium(F12K) supplemented with fetal bovine serum 5%, L-glutamine,streptomycin, amphotericin B and sodium bicarbonate. All cell lines weremaintained at 37° C., 5% CO₂ in a humidified incubator. MCF-7 cells weregrown in Eagle's Minimal Essential medium supplemented with fetal bovineserum (10%) with 0.01 mg/mL bovine insulin and 1% penicillin.

Cellular Uptake of Polymer-Coated Nanoceria [DNC (0), PNC (−) and ANC(+)]

Ten thousand (10,000) cells including cardiomyocytes, human embryonickidney cells, lung carcinoma cells and breast carcinoma cells that wereseeded in petri dishes and incubated with DiI-labeled-DNC (0),DiI-labeled-PNC (−) and DiI-labeled-ANC (+) (1.0 mM), for 3 h at 37° C.,5% CO₂ in a humidified incubator.

Then, the cells were washed with 1× PBS and fixed with 10% formalin in1× PBS. Afterwards, the cells were incubated with DAPI (1 mg/mL,Molecular Probes) for 10 minutes. Then the cells were washed andvisualized under a confocal microscope (Zeiss LSM 510).

Inhibition of Cellular Uptake of the Polymer-Coated Nanoceria

For these studies, 10,000 cells (H9c2 and A549 ) were treated with theinhibitors sodium azide (10 mM) and 2-dexoyglucose (50 mM) for 30minutes. Then, the cells were incubated with DiI-labeled PNC (−) andDiI-labeled ANC (+) preparations (1.0 mM) for 3 h at 37° C., 5% CO₂ in ahumidified incubator. Fixation and subsequent staining with DAPI wereperformed as described above. Experiments were done at 4° C. incubatingthe cells with the DiI-labeled PNC (−) and DiI-labeled ANC (+)prepartions (1.0 mM) for 3 h.

Lysosomal Staining

After treatment of the corresponding cell lines with DiI-labeled-DNC(0), DiI-labeled-PNC (−) and DiI-labeled-ANC (+) for 3 hours, cells werewashed and incubated for 20 minutes with Lysotracker (Invitrogen) (35nM) at 37° C., 5% CO₂ in a humidified incubator. Fixation procedureswere performed as stated before.

Lysosomal Isolation and Oxidase-Activity Determination of EntrappedNanoceria

Lysosome were isolated using a previously reported procedure describedby Schroter et al., in A Rapid Method to Separate Endosomes fromLysosomal Contents Using Differential Centrifugation and Hypotonic Lysisof Lysosomes. J Immunol Methods 1999, 227, 161-168. Ten thousand(10,000) cells (cardiomyocytes, human embryonic kidney cells, lungcarcinoma cells and breast carcinoma cells) were seeded in petri dishesand incubated with ANC (+), PNC (−) and DNC (0) (1.0 mM) for 3 h at 37°C., 5% CO₂ in a humidified incubator.

Then, the cells were washed with 1× PBS, trypsinized and centrifuged at1,000 rpm for 8 minutes. In order to isolate the lysosomes, cells wereresuspended in an isotonic sucrose solution (1.0 mL, 0.08 M CaCl₂, 0.25M sucrose, 10 mM Tris-HCl) to lyse the cells into cytosolic andorganelles fractions and centrifuged at 25,000 g for 15 minutes.

Subsequently, the supernatant was carefully removed, and the pellet wasresuspended in 1.0 mL 150 mM KCl (10 mM Tris-HCl, pH 7.4) followed bycentrifugation at 25,000 g for 15 minutes to sediment the lysosomes. Thepellet containing the lysosomes was resuspended in 200 μL of TMB (1.0mg/mL) and incubated overnight at room temperature.

After the incubation period, the absorbance at 652 nm of the lysosomesuspension was recorded.

Cell Viability Assays

Cells (cardiomyocytes, human embryonic kidney cells, lung carcinomacells and breast carcinoma cells) were seeded in 96-well plates at adensity of 3,000 cells per well and incubated with ANC (+), PNC (−) andDNC (0) (1.0 mM) for 3 h. Then, 0.5 mM of MTT[3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (Sigma)was added, followed by incubation for 24 h. After 24 h, the resultingcrystals were dissolved in 40 μL of acidified isopropanol and theabsorbance at 570 nm was recorded using a plate reader (Bio-TEK, SynergyHT Multidetection Microplate reader).

Discussion of Research

The toxicity of nanomaterials depends on various factors, including thenature and chemical composition of the nanoparticle's core, mode ofsynthesis, size, shape and crystallinity, surface reactivity, solubilityin aqueous media and degree of aggregation. Although efforts have beenmade to study the effect of the polymeric surface coating and surfacecharge on the uptake, localization and toxicity of nanomaterials, themechanistic implications are still not completely understood. This ishighly important as some nanomaterials, such as cerium oxide, maydisplay either a beneficial (antioxidant) or toxic (oxidant) effect,depending on the pH of the compartment where they localize inside thecell. Here, we have shown that polymer-coated nanoceria displaysdifferent levels of toxicity, depending upon cellular uptake andsubsequent subcellular localization. We found that when polymer-coatednanoceria internalize and localize in the lysosomes, it becomes toxicdue to the acidic microenvironment of these organelles, which activatesthe oxidase activity of nanoceria as shown in FIG. 11.

However, lower toxicity is observed when polymer-coated nanocerialocalize into the cytoplasm of cells and when is not internalized by thecells. Our results also show that the internalization, eventuallocalization and cytotoxicity of polymer-coated nanoceria within thecell greatly depend on the surface charge of the polymeric coating andthe type of cell (cancerous versus normal). For instance, we have foundthat the aminated nanoceria [ANC(+)] were toxic to most of the cancerand normal cell lines studied, as these nanoparticles were uptaken andlocalized mostly into the lysosomes. This might indicate thatnanoceria's intrinsic oxidant behaviour at acidic pH is responsible forthe observed cytotoxic of the cationic polymer-coated nanoparticles[ANC(+)]. Meanwhile, PNC(−) internalized mostly into the A549 lungcarcinoma cells but not significantly into any of the other cell linesstudied. This observation is corroborated by the fact that no lysosomallocalization and therefore no toxicity is observed in MCF-7, H9c2 orHEK293 cells treated with PNC(−).

In contrast, significant lysosomal localization and cytotoxicity isobserved in the A549 cells treated with PNC(−). Surprisingly, dintinctchanges in cellular uptake were observed with the dextran polymer-coatedcerium oxide nanoparticles [DNC(0)]. A very disperse and diffusedintracellular distribution was seen in all cell lines (cancer andnormal) exposed to DNC (0), with very few of the DNC(0) localizing intothe lysosomes. Therefore, the neutral DNC (0) nanoparticles were foundto be non toxic to any of the cell lines studied as they localizeprimarily in the cytoplasm. This suggests that the observed non-toxicbehavior of DNC(0) might be attributed to its significantly lowentrapment into lysosomes, contrary to ANC(+). Based on these results,DNC(0) would be a great platform for further developmentof therapeuticantioxidant nanoagents, as they exhibit minimal toxicity. In addtion,they can be effectively used in radiation therapy, since they are nottoxic to normal cells. Also, as dextran-coated nanoparticles have beenfound to have long circulation time, similarly dextran coated ceriumoxide nanoparticles can be employed as long circulating antioxidantnanoagents.

Another factor, which may contribute to the cytotoxic behavior ofpolymer-coated nanoceria is the type of cell line used and pH of thecell's microenvironment as reported by M. C. Brahimi-Horn et al., inHypoxia Signalling Controls Metabolic Demand. Curr Opin Cell Biol 2007,19, 223-229; J. W. Kim et al., in Cancer's Molecular Sweet Tooth and theWarburg Effect. Cancer Res 2006, 66, 8927-8930; P. M. Smith-Jones etal., in Early Tumor Response to Hsp90 Therapy Using HER2 PET: Comparisonwith 18F-FDG PET. J Nucl Med 2006, 47, 793-796; and M. Wu et al., inMultiparameter Metabolic Analysis Reveals a Close Link BetweenAttenuated Mitochondrial Bioenergetic Function and Enhanced GlycolysisDependency in Human Tumor Cells. Am J Physiol Cell Physiol 2007, 292,C125-136.

The factors that contribute to the cytotoxic behavior of polymer-coatednanoceria are particularly relevant because tumor progression, increasedinvasion, metastasis and acidic tumor environment have been found to beinterrelated, as discussed by R. J. Gillies in The TumorMicroenvironment: Causes and Consequences of Hypoxia and Acidity.Introduction. Novartis Found Symp 2001, 240, 1-6; R. J. Gillies et al.,in Frontiers in the Measurement of Cell and Tissue pH. Novartis FoundSymp 2001, 240, 7-19; discussion 19-22, 152-153; N. Raghunand et al., inpH and Chemotherapy. Novartis Found Symp 2001, 240, 199-211; discussion265-198; N. Raghunand et al., in Acute Metabolic Alkalosis EnhancesResponse of C3H Mouse Mammary Tumors to the Weak Base Mitoxantrone.Neoplasia 2001, 3, 227-235; E. K. Rofstad et al., in AcidicExtracellular pH Promotes Experimental Metastasis of Human MelanomaCells in Athymic Nude Mice, Cancer Res 2006, 66, 6699-6707; and M.Stubbs et al., in Causes and Consequences of Acidic pH in Tumors: AMagnetic Resonance Study. Adv Enzyme Regul 1999, 39, 13-30.

Differences between normal and tumor tissue and tumor-to-tumor variationmay play a key role in dictating the antioxidant vs oxidase behavior ofthe polymer-coated nanoceria. Previously, we have reported thatpolymer-coated nanoceria displays unique oxidase-like behavior atslightly acidic pH. Therefore, in view of this oxidase-like property atacidic pH, intracellular distribution of the polymer-coated nanoceriainto the lung carcinoma's lysosomes may be the reason for thesenanoparticles cytotoxicity. In addition, as A549 lung carcinoma cellsand most tumors have been found to have upregulated glycolysis andincreased lactic acid production, this effect might further contributetowards the buildup of an acidic microenvironment, favoring nanoceria'soxidase activity and therefore sensitizing tumors towards radiationtherapy.

In summary, the role of the surface-coated nanoceria and resultingsurface charge influences cell internalization, subcellular distributionand differential antioxidant/oxidant activity shedding new light indelineating the mechanisms that lead to the toxicity or non-toxicproperties of nanoceria. A surface-coating that introduces a positive,negative or neutral charge to nanoceria, controls the nanoparticletoxicity.

It has been illustrated, that dextran coated nanoceria (DNC), apolysaccharide, provides a neutral surface charge to nanoceria, and itis always non-toxic. Thus, DNC creates a nontoxic nanoceria that eitherdoes not become internalized into normal cells or transformed cells, orif cellular uptake occurs, the DNC particle goes into the cytoplasmwhere it is not toxic.

With regard to polyacrylic acid coated nanoceria (PNC), a carboxylatedpolymer, provides a negative surface charge to nanoceria, and is toxicin lung carcinoma cells (A549) when subcellular localization occurs inthe lysosomes, but is non-toxic in other transformed and non-transformedcells (MCF-7, H9c2 or HEK293) when cellular uptake does not occur orsubcellular localization occurs in the cytoplasm.

Aminated polyacrylic acid coated nanoceria (ANC), which results from theconjugation of ethylenediamine with polyacrylic acid, is an example of acoating that provides a positive surface charge to the nanoceria, and itis toxic in normal and transformed cells (A549, H9c2 or HEK293) whensubcellular localization occurs in the lysosomes, but is non-toxic inother transformed cells, such as breast carcinoma cells (MCF-7) whencellular uptake does not occur or subcellular localization occurs in thecytoplasm.

Thus, polymer-coated nanoceria with different surface charges, such as,positive, negative and neutral, display discrete subcellularlocalization and toxicity profiles. No toxicity was observed whennanoceria localized to the cytoplasm of cells, however, uponlocalization to the lysosomes, the low pH of this organelle activatesthe oxidase-like activity of the nanoparticle, exhibiting toxicity. Thenanoceria intracellular localization is dependent on the nanoparticlesurface charge and the type of cell.

While the invention has been described, disclosed, illustrated and shownin various terms of certain embodiments or modifications which it haspresumed in practice, the scope of the invention is not intended to be,nor should it be deemed to be, limited thereby and such othermodifications or embodiments as may be suggested by the teachings hereinare particularly reserved especially as they fall within the breadth andscope of the claims here appended.

We claim:
 1. A kit for treating cancer, the kit comprising a quantity ofcerium oxide nanoparticles coated with a polymer providing a surfacecharge, and at least one medical application device, wherein saidmedical application device and said nanoparticles are packaged together.2. The kit of claim 1, wherein the surface charge is a negative surfacecharge.
 3. The kit of claim 2, wherein said nanoparticles are coatedwith polyacrylic acid.
 4. The kit of claim 1, wherein the medicalapplication device is a syringe.
 5. The kit of claim 1, wherein themedical application device is a pill.